lipoteichoic-acid and Peritonitis

Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Cell Line; Enterococcus faecalis; Female; Gram-Positive Bacterial Infections; Host-Pathogen Interactions; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Microbial Viability; Molecular Sequence Data; Myeloid Differentiation Factor 88; NF-kappa B; Peritonitis; Phagocytosis; Protein Structure, Tertiary; Signal Transduction; Teichoic Acids

Type 1 lipoteichoic acid (LTA) is present in many clinically important gram-positive bacteria, including enterococci, streptococci, and staphylococci, and antibodies against LTA have been shown to opsonize nonencapsulated Enterococcus faecalis strains. In the present study, we show that antibodies against E. faecalis LTA also bind to type 1 LTA from other gram-positive species and opsonized Staphylocccus epidermidis and Staphylcoccus aureus strains as well as group B streptococci. Inhibition studies using teichoic acid oligomers indicated that cross-reactive opsonic antibodies bind to the teichoic acid backbone. Passive immunization with rabbit antibodies against E. faecalis LTA promoted the clearance of bacteremia by E. faecalis and S. epidermidis in mice. Furthermore, passive protection also reduced mortality in a murine S. aureus peritonitis model. The effectiveness of rabbit antibody against LTA suggests that this conserved bacterial structure could function as a single vaccine antigen that targets multiple gram-positive pathogens.

Topics: Adult; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacteremia; Disease Models, Animal; Enterococcus faecalis; Female; Glycerophosphates; Human Experimentation; Humans; Immunization, Passive; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Opsonin Proteins; Peritonitis; Phagocytosis; Rabbits; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Survival Analysis; Teichoic Acids

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naïve WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

Topics: Animals; Cell Movement; Chemotaxis; Down-Regulation; G-Protein-Coupled Receptor Kinase 2; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Mice; Neutrophils; Peritonitis; Receptors, Interleukin-8B; Sepsis; Signal Transduction; Survival Analysis; Teichoic Acids; Toll-Like Receptor 2

There are no predictive factors for peritoneal dialysis-associated peritonitis; however, its resolution correlates with a cell-mediated Th1 immune response. We tested the hypothesis that induction of receptor-interacting protein 2 (RIP2), an assumed kinase linked with Th1 responses, is a useful marker in this clinical setting. Basal RIP2 expression was measured in human immune cells and during dialysis-associated peritonitis. RIP2 increased with bacterial toxin cell activation and the temporal profile for this differed depending on immune cell involvement in the innate or adaptive phases of the response. Importantly, RIP2 expression increased in peritoneal immune cells during dialysis-associated peritonitis and this upregulation correlated with clinical outcome. An early induction in peritoneal CD14(+) cells correlated with rapid resolution, whereas minimal induction correlated with protracted infection and with catheter loss in 36% of patients. These latter patients had higher levels of MCP-1 consistent with a delayed transition from innate to adaptive immunity. Our study shows that upregulation of RIP2 is a useful marker to monitor dialysis-associated peritonitis and in predicting the clinical outcome of these infections.

Topics: Adult; Aged; Biomarkers; Female; Humans; Interleukin-12 Subunit p35; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Lipopolysaccharides; Male; Middle Aged; Peptidoglycan; Peritoneal Dialysis; Peritonitis; Receptor-Interacting Protein Serine-Threonine Kinase 2; RNA, Messenger; Teichoic Acids; Tetradecanoylphorbol Acetate; Toll-Like Receptor 2; Toll-Like Receptor 4; Up-Regulation

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2(-/-) or C5aR(-/-) mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2(-/-) mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.

Topics: Animals; Antigens, Bacterial; Chemotaxis, Leukocyte; Complement C5a; Disease Models, Animal; E-Selectin; Ligands; Lipopolysaccharides; Macrophages; Mast Cells; Mice; P-Selectin; Peptidoglycan; Peritonitis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4

The adherence of staphylococci to monolayers of human mesothelial cells was studied. Adherence of Staphylococcus aureus to mesothelial cell monolayers was 3.4-fold better than to plastic (P less than .01) whereas that of Staphylococcus epidermidis was 3.0-fold less than to plastic (P less than .01). Neither serum albumin nor gelatin inhibited staphylococcal binding. S. aureus adherence correlated with the amount of cell wall protein A (r = .63, P less than .05) but not with fibronectin binding; it was significantly inhibited by the addition of purified cell wall lipoteichoic acid (55% +/- 2.7%), teichoic acid (34.5% +/- 3.4%), and protein A (25.6% +/- 2.9%) but not peptidoglycan. Protein A- and teichoic acid-deficient mutants adhered less well than their parent strains, and encapsulated S. epidermidis adhere well to human monothelial cells. Staphylococcal binding may involve cell wall lipoteichoic acid, teichoic acid, and protein A.

Topics: Bacterial Adhesion; Blood Proteins; Cell Wall; Cells, Cultured; Humans; Lipopolysaccharides; Peptidoglycan; Peritoneum; Peritonitis; Staphylococcal Protein A; Staphylococcus aureus; Staphylococcus epidermidis; Teichoic Acids